determinedzhuo爱

CARDIOVASCULARPHYSIOLOGYDelayedconductionanditsimplicationsinmurineScn5a+/hearts:independentandinteractingeffectsofgenotype,age,andsexKamalanJeevaratnam&SuiPohTee&YanminZhang&RebeccaRewbury&LailaGuzadhur&RudolfDuehmke&AndrewA.
Grace&MingLei&ChristopherL.
-H.
HuangReceived:1July2010/Revised:29October2010/Accepted:12November2010/Publishedonline:3December2010#TheAuthor(s)2010.
ThisarticleispublishedwithopenaccessatSpringerlink.
comAbstractWeexploredforrelationshipsbetweenSCN5Ahaploinsufficiency,implicatedinclinicalarrhythmogenic-ity,andrightventricular(RV)conductiondisordersinLangendorff-perfused,maleandfemale,andyoung(3months)andold(>12monthold)Scn5a+/andwildtype(WT)hearts.
Theinvestigatedconditionsofgenotype,age,andsexaffectedlatenciesbutnotrepolarizationtimecoursesofRVmonophasicactionpotentials.
ThispromptedexaminationofthepatternsofRVepicardialactivation,itsdispersion,andtheirinterrelationshipsaspossiblearrhyth-micmechanismsusinga64-channel,multi-electrodearray.
Meanventricularactivationtimes(T*MEAN),spatialdis-persions(D*S)betweenrecordingchannels/cardiaccycle,andmaximumactivationtimes(T*MAX)representingtheslowestpossibleconductioninanygivenheartwereallhigherinoldmaleScn5a+/comparedwithyoungmaleandoldfemaleScn5a+/andoldmaleWT.
Temporaldis-persions(D*T)ofrecordingchannelsweresimilarlyhigherinoldmaleScn5a+/comparedwitholdmaleWT.
AllgroupingsofD*T,D*S,andT*MAXneverthelesslinearlycorrelatedwithT*MEAN,withindistinguishableslopes.
ThevariatesexploredthusinfluenceD*T,D*S,andT*MAXthroughactionsonT*MEAN.
Thesefindingsinturncorrelatedwithincreasedlevelsoffibrosisinyoungmale,youngfemale,andoldmaleScn5a+/comparedwiththecorrespondingWTs.
Wethusdemonstrateforthefirsttimeindependentandinteractingeffectsofgenotype,age,andsexonepicardialconductionanditsdispersionsatleastpartiallyattributabletofibroticchange,resultinginthegreatesteffectsinoldmaleScn5a+/inanabsenceofalterationsinrepolarizationtimecourses.
Thisdirectlyimplicatesaltereddepolarizationintheclinicalarrhythmo-genicityassociatedwithScn5a+/.
KeywordsMulti-array.
Biophysics.
Conduction.
Dispersion.
Depolarization.
RepolarizationIntroductionTherehasbeenincreasinginterestinthepossiblephysio-logicalimplicationsofhaploinsufficienciesinvolvingK.
Jeevaratnam(*):S.
PohTee:Y.
Zhang:R.
Rewbury:L.
Guzadhur:R.
Duehmke:C.
L.
-H.
HuangPhysiologicalLaboratory,UniversityofCambridge,DowningStreet,CambridgeCB23EG,UKe-mail:drkamalanjeeva@gmail.
comK.
Jeevaratnam:L.
Guzadhur:R.
Duehmke:A.
A.
Grace:C.
L.
-H.
HuangCardiovascularResearch,DepartmentofBiochemistry,HopkinsBuilding,UniversityofCambridge,CambridgeCB21QW,UKM.
LeiCardiovascularGroup,SchoolofClinical&LaboratorySciences,UniversityofManchesterCoreTechnologyFacility(3rdfloor),UniversityofManchester,ManchesterM139NT,UKY.
ZhangDepartmentofPaediatrics,FirstAffiliatedHospital,CardiovascularIonChannelDiseaseLaboratory,Xi'anJiaotongUniversity,Xi'an710061,People'sRepublicofChinaK.
JeevaratnamDepartmentofHumanBiology,FacultyofMedicine,InternationalMedicalUniversity,BukitJalil,57000,KualaLumpur,MalaysiaPflugersArch-EurJPhysiol(2011)461:29–44DOI10.
1007/s00424-010-0906-1voltage-gatedNa+channelsforclinicalcardiacelectrophys-iologicaldisorders.
ThelatterincludetheBrugadasyn-drome(BrS)andprogressivecardiacconductiondisease(PCCD),bothofwhichareassociatedwitharrhythmicandconductionabnormalities[8].
Pathophysiologicalfindingshaveimplicatedtherightventricle(RV)particularlytherightventricularoutflowtractastheprimarylocationforelectricaldisordersrelatedtoBrS[4].
BrStypicallypresentsassyncopeandcardiacarresttypicallyoccurringinthethirdorfourthdecadeoflifeinmalesratherthanfemales[14].
Thus,menwithBrShaveahigherprevalenceoflife-threateningarrhythmiasthanwomencorrespondingtoincidencesof9.
5%and3.
8%,respectively[18].
BrSisalsotheleadingcauseofdeathofmenundertheageof50yearsinendemicregions[2].
EightypercentofpatientsinWesternpopulationsand90%inAsiancountriesshowingclinicalmanifestationsofBrScausedbySCN5Amutationaremales[22].
ThecorrespondingincidenceshavenotbeenestablishedforPCCD.
GeneticallymodifiedmurinesystemshavebeenusefulinexploringtheconsequencesofSCN5Ahaploinsufficiencies.
Theypotentiallyfullyreplicatethegeneticchangesassoci-atedwitharrhythmogenicconditionswithoutrequiringpharmacologicalmanipulation.
Langendorff-perfusedScn5a+/heartsweremorearrhythmicthantheirwildtype(WT)counterparts[14,23].
Inaddition,ageandsexinfluencedmurineScn5a+/conductionphenotypes.
Scn5a+/miceshowed~20%increasesinthedurationsoftheirQRScomplexes[17].
Morerecentdetailedelectrocar-diographic(ECG)studiesdemonstratedindependentandinteractingeffectsofageandsexonbothsino-atrialandatrioventricularfunction,consistentwithmarkedconductionalterationsparticularlyinoldmaleScn5a+/thatparalleledclinicalarrhythmogenicfindings[7].
ElectrophysiologicalchangesinBrShavebeenreplicat-edinarteriallyperfusedcaninerightventricularwedgesthroughpharmacologicalmanipulation.
SuchexperimentsdescribedlocalizedlossesoftheAPdomewithpotentialarrhythmogeniceffects[1,5,26].
RVconductionblockhasalsobeenimplicatedinthedevelopmentofarrhythmiasinBrS[9,12].
ArecenthumanclinicaltrialonBrSpatientsattributedtheirtype1ECGpatternstolocaldepolarizationasopposedtorepolarizationabnormalities[15].
MurineScn5a+/micesimilarlydemonstratedprolongedECGPwaveandPRdurations,rightaxisdeviation,andsecond-degreeAVblocksuggestingconductiondeficiencies[14].
Theirelectrogramdurations(EGDs)duringbipolarelectrogramrecordingsparticularlyfollowingprematurestimulationwerealsoincreased[23].
BoththeseabnormalpatternsofmyocardialactivationandchangesinEGDwereattributedtore-entrantsubstratesresultingfromaltered("fractionated")conductionvelocitydistributions[19–21,24].
ThepresentstudyexploresthehypothesisthatScn5a+/genotype,age,andsexaltercardiacconduction.
Thisinturnwouldalterthedispersionsintheresultingmyocardialactivationtimesatthetissuelevel,therebyinfluencingarrhythmictendency.
Theelectrophysiologicalexperimentscombinedmonophasicactionpotentialandmulti-electrodearrayrecordings.
Theystudiedactionpotentialproperties,followedbydetailedanalysesofepicardialactivationtimesandtheirdispersionforthefirsttime,forsubsequentcomparisonwithmorphologicalstudiesexploringforpossiblerolesoftissuefibrosisinsuchchanges.
Theobservedvariationsinactivationpropertieswithgenotype,age,andsexdirectlycorrelatedwiththecorrespondingfibrosis,suggestingamechanismforsuchconductionchanges.
Theseinturndirectlyaccountedforthepreviouslyreportedclinicaldifferencesincardiacarrhythmogenicity.
MethodsExperimentalgroupsMicewerehousedinananimalfacilityat21°Cwith12-hlight/darkcycles.
Animalswerefedsterilechow(RM3MaintenanceDiet,SDS,Witham,Essex,UK)andhadfreeaccesstowater.
AllprocedurescompliedwiththeUKHomeOfficeregulations(Animal(ScientificProcedures)Act1986).
Inordertodevelopacomprehensiveriskassessmentmodelofthetwotypesofmicestudied,atotalof23WTand21Scn5a+/knockoutmicewereused.
Allmiceusedforthisstudywerederivedfromtheirrespective129/svbackgroundstrainstoavoidanypossiblestrainrelatedvariationinthestudy.
Themiceweredividedintoeightgroups.
Group1wasWTmaleaged3months(n=7),group2wasWTmaleaged>12months(n=5),group3wasWTfemaleaged3months(n=6),group4wasWTfemaleaged>12months(n=5),group5wasScn5a+/maleaged3months(n=6),group6wasScn5a+/maleaged>12months(n=5),group7wasScn5a+/femaleaged3months(n=5),andgroup8wasScn5a+/femaleaged>12months(n=5).
ExperimentsusedaLangendorff-perfusedpreparationadaptedforthemurineheart,asdescribedpreviously[6,23].
Allassessmentswereper-formedfullyblinded,andcodeswerebrokenonlyafterallmeasurementshadbeenperformed.
Multi-electrodearrayrecordingsAcustom-madeelectrodearrayconsistingof64separateelectrodes(Teflon-coatedsilverwires;0.
125mmdiameter;ScienceProducts)inan8*8configurationwithaninter-electrodedistanceof0.
55mmwasused.
Unipolarrecord-ingswereperformedbyplacingthemulti-electrodearrayontherightepicardiumwiththereferenceelectrode,actingas30PflugersArch-EurJPhysiol(2011)461:29–44theindifferentpoleatthebaseoftheheart.
The64recordingelectrodeswereconnectedthroughshieldedwirestotwo32-channelamplifiers(SCXI-1102C,NationalInstrumentsCorporation(U.
K.
)Ltd,Newbury,UK).
Sam-plingfrequencyforeachsignalwassetat1kHz.
Thesignalswerecontinuouslystoredondiskanddisplayedonscreenusingacustom-developedprogram,writteninLabview7.
0(NationalInstrumentsCorporation(U.
K.
)Ltd,Newbury,UK).
Fortheoff-lineanalysis,signalsweredisplayedonscreeninsetsofeightto16electrograms.
Theactivationtimewasdeterminedasthepointofmaximalnegativeslopeandmarkedwithacursor.
Aftermarkingallsignificantwaveformsinallleads,theactivationtimeswerethendisplayedinagridrepresentingthelayoutoftheoriginalrecordingarray.
Allactivationtimes,inmilliseconds,wererelatedtothetimingofthefirstdetectedwaveform.
ActivationmapsweredrawnusingMicrosoftOfficeExcel2007(MicrosoftCorporation,SiliconValley,USA).
MonophasicactionpotentialrecordingsAbipolarplatinumstimulatingelectrode(1-mminterpolarspacing)wasplacedontheinterventricularseptum,andamonophasicactionpotential(MAP)recordingelectrode(HugoSachs,HarvardApparatus)wasplacedonthecorrespondingrightepicardium.
Duringthistime,MAPsshowedstablebaselines,rapidupstrokephasesthatreachedconsistentamplitudes,andsmoothrepolarizationphases,allofwhichareknowntobecharacteristicofhigh-fidelityMAPrecordings.
MAPswereamplified,band-passfiltered(0.
5Hz–1kHz:NeurologNL104Amplifer,NL125/6Filter;Digitimer,WelwynGardenCity,Herts,UK)anddigitizedatasamplingfrequencyof5kHz(micro1401,CambridgeElectronicDesign,Cambridge,UK).
Heartswerepacedatacyclelengthof125ms.
AnalysisofMAPswasperformedusingSpikeIIsoftware(CambridgeElectronicDesign).
QuantificationofcardiacfibrosisIsolatedheartswereflushedwithKrebsbufferandperfusedwith4%bufferedformalinfor5minbeforebeingimmersedinformalinovernight.
Afterfixation,grosstransversesectionswerecutfrombasetoapex,andtheheartsweresubjectedtoroutinetissueprocessingandparaffinembedding.
Paraffinsectionsof7μmthicknesswerethencutandstainedwithSiriusredstain(Sigma-Aldrich,Dorset,UK).
Allsectionsweresubsequentlyviewed,magnified,anddigitallyacquiredusingtheNanoZoomer2.
0DigitalPathologysystem(Hamamatsu,Hertfordshire,UK).
Forthequantificationoffibrosis,tworandomlyselectedphotomicrographswithaminimumdistanceof14μmfromeachotherweretakenforeveryheart.
Followinga*2magnification,acustom-made17cm*23cmmorphometricgrid,consistingofsquareboxesofdimension1cm*1cm,correspondingtoa0.
26mm*0.
26mmareaoftissue,wasthensuperimposedoneachphotomicrograph.
Onlysquaresoccupiedeithercompletelyorpartiallybycardiactissueswerecounted.
Similarly,squarescontainingfibroticchangeswerethencounted,andthiswasexpressedasapercentageofthetotalcardiactissuearea.
Anaveragepercentageoffibroticchangesforeveryhearttakenfromthetwophotomicrographswereobtainedandthenaccumulatedforeverygrouptoprovidethemeanpercentageoffibrotictissue.
StatisticalanalysisComparisonswhereappropriatewereperformedbyanalysisofvarianceorttestwithSPSSsoftware(SPSSUK,Woking,Surrey,UK).
Regressioncoefficientswerecalculatedbyt=r√(n2)/√(1r2),withn2degreesoffreedom[16].
StatisticalsignificancewasassumedatP10ms,inordertoemphasizegroupsshowingeitherthegreatestorsmallestextremesinsuchvalues.
Thesesuccessivestratificationsdemonstratedsignificantinde-pendentandinteractingeffectsofgenotype,age,andsex.
Thus,eventhesimpleinitial,genotypic,comparisonofthepopulationasawholedemonstratedsignificantlyhigher(P=0.
002)T*MAXvaluesintheScn5a+/comparedwiththeWT.
Scn5a+/andWTcorrespondinglyshoweddistributionsbiasedtowardgreaterandsmallerTMAXvalues,respectively.
Furtherstratificationofthedatasetbybothgenotypeandagedemonstrateddifferenteffectsofagewithineachgenotype.
Thus,therewerestatisticallyindistinguishableandsimilarlydistributedT*MAXvaluesinoldWTandyoungWThearts.
However,suchT*MAXvaluesweresignificantlygreater(P=0.
031)inoldScn5a+/thanyoungScn5a+/,reflectingdistributionsskewedinoldScn5a+/towardshighervalues,>10ms,ofTMAX.
Incontrast,analysisoftheeffectsofgenotypewithineachagegroupdemonstratedsignificantlyhigher(P=0.
046)T*MAXvaluesintheyoungScn5a+/heartscomparedwiththeyoungWT.
Similarly,T*MAXvaluesweresignificantlyhigher(P=0.
016)intheoldScn5a+/comparedwitholdWT.
Stratificationsbybothgenotypeandsexrevealedthattherewerenodifferencesattributabletosexwithineachgenotype.
Thus,T*MAXvaluesweresimilarbothbetweenmaleandfemaleWTandbetweenmaleandfemaleScn5a+/.
However,comparisonsoftheeffectsofgenotypewithineachsexgroupshowedsignificantlyhigher(P=0.
007)T*MAXvaluesinthemaleScn5a+/thanthemaleWT.
Incontrast,T*MAXvalueswerestatisticallyindistinguishablebetweenfemaleWTandfemaleScn5a+/.
Table4D*S(ms)valuesinrightventricularmurineheartsAnimalSamplesAverage±SEWT(total)231.
61±0.
18WTyoung131.
51±0.
21WTold101.
74±0.
32WTmale121.
55±0.
29WTfemale111.
68±0.
21WTyoungmale71.
65±0.
31WTyoungfemale61.
35±0.
28WToldmale51.
41±0.
59aWToldfemale52.
07±0.
22Scn5a+/(total)211.
99±0.
24Scn5a+/young111.
57±0.
25Scn5a+/old102.
46±0.
36Scn5a+/male112.
40±0.
40Scn5a+/female101.
55±0.
14Scn5a+/youngmale61.
52±0.
47bScn5a+/youngfemale51.
62±0.
13Scn5a+/oldmale53.
44±0.
24a,b,cScn5a+/oldfemale51.
48±0.
27cMeanswithsimilarlowercaselettersdiffersignificantlyfromeachotheratP10msincomparisonwiththerestofthegroup.
Incontrast,therewerenoothersignificantdifferencesinT*MAXvaluesamongtheremain-inggroups.
CorrelationsbetweenactivationtimesandmeasuresoftheirdispersionTheaboveresultsdemonstratesignificantinteractingeffectsofage,sex,andgenotypeuponmeasuresofactivationtimes,T*MEAN,itsdispersions,asrepresentedbyD*T,D*S,andT*MAX.
Thus,inthegroupasawhole,genotypeexertedsignificanteffectsuponT*MEAN,D*T,andT*MAX.
StratificationbygenotypeandageinfluencedonlytheT*MAXvaluesandthatbygenotypeandsexinfluencedT*MEAN,D*T,andT*MAX.
Thefinalstratificationsrevealedeffectsonallfourvariables.
Allsignificantchangestendedtooccurinthesamedirection,consistentwithcausalrelationshipsbetweenthem.
Furthermore,significantchangesinD*TandD*SwerealwaysaccompaniedbycorrespondingchangesinT*MEAN,butthereversewasnotalwaysthecase.
Together,thesefindingssuggestatrendtowardincreaseddispersionsofactivationwithincreasingactivationtime,establishingthelattervariantasamajordeterminantofsuchdispersion.
ThesefeaturespromptedustoexploreforquantitativerelationshipsbetweenthemeasuresofdispersionwiththeircorrespondingT*MEAN.
Firstly,weaccordinglycomparedD*T,D*S,andT*MAXvaluesfromeachindividualheartagainsttheircorrespondingT*MEAN.
Figure4exemplifiestheresultingAnimalSamplesAverage±SEIntervals0–3ms4–6ms7–10ms>10msWT(total)235.
87±0.
65a8681WTyoung135.
31±0.
73b5440WTold106.
60±1.
17c3241WTmale125.
33±1.
03d5421WTfemale116.
46±0.
793260WTyoungmale75.
29±0.
992320WTyoungfemale65.
33±1.
173120WToldmale55.
40±2.
21e3101WToldfemale57.
80±0.
740140Scn5a+/(total)219.
29±0.
86a0696Scn5a+/young117.
46±0.
62b,f0461Scn5a+/old1011.
30±1.
44c,f0235Scn5a+/male1110.
46±1.
48d0326Scn5a+/female108.
00±0.
620370Scn5a+/youngmale66.
83±1.
01g0321Scn5a+/youngfemale58.
20±0.
580140Scn5a+/oldmale514.
80±1.
36e,g,h0005Scn5a+/oldfemale57.
80±1.
16h0230Table5T*MAX(ms)andfre-quenciesofheartswithdifferentrangesofTMAXvaluesMeanswithsimilarlowercaselettersdiffersignificantlyfromeachotheratP0.
05).
ComparisonsacrossgenotypicgroupdemonstratedmorefibrosisinyoungmaleScn5a+/,youngfemaleScn5a+/andoldmaleScn5a+/comparedwiththeyoungmaleWT,youngfemaleWT,andoldmaleWT(P=0.
001,P=0.
002,andP22-mscutoffsintheirQRSdurations,inparallelwithsimilarclinicalstratificationsshown(a)WTYoungMale(b)WTYoungFemale(c)WTOldMale(d)WTOldFemale3mm(e)Scn5a+/-YoungMale(f)Scn5a+/-YoungFemale(g)Scn5a+/-OldMale(h)Scn5a+/-OldFemale3mmFig.
5RepresentativeslideforcardiacfibrosisstaininginWTandScn5a+/heartsgroupedbyageandsex.
Thestudypopula-tionwasstratifiedinto:WTyoungmale(a),WTyoungfemale(b),WToldmale(c),WToldfemale(d),Scn5a+/youngmale(e),Scn5a+/youngfemale(f),Scn5a+/oldmale(g),andScn5a+/oldfemale(h).
HeartswereroutinelystainedwithSiriusred,andmorpho-metricanalysisforpercentageoffibrosiswasperformedforalleightgroups.
Areasofincreasedreduptakesignifypresenceoffibroticchanges.
Horizontalbarbelowsectionsineachpaneldenotesa3mmdistance40PflugersArch-EurJPhysiol(2011)461:29–44byBrSpatientscontainingSCN5Amutations[10].
Thesedemonstratedparallelchangesinconductionandinsemi-quantitativemeasuresoffibrosisthatvariedwithage,confirmingpreviouscorrelationsbetweenincreasedPRintervalsandagespecificallyintheScn5a+/[17].
Oursubsequentstudyusingcloselysimilarsamplenumbersthendescribedconductionvelocityalterationsassociatedwithincreasedcardiacfibrosisanddown-regulationofconnexinexpressioninoldScn5a+/mice.
WethusdemonstratedthatsuchalterationsinconductionvelocitywerenotsolelydependentonreducedScn5aexpression.
Wethenhoweverdidnotstratifyfortheinteractingeffectsofageandsex[25].
Finally,recentECGmeasurementsparticularlyofPRandRRintervalsthenwentontofullyclarifyboththeindependentandtheinteractingeffectsofgenotype,age,andsexuponconductioncharacteristicsinfullystratifiedexperimentalgroups.
TheseshowedinparticularthatoldmaleScn5a+/showedmarkedincreasesinsuchconductiondurations[7].
Weproceededtoexaminemeanconductionanditsdispersionatthetissue,ratherthantheECGlevel,usingmulti-arrayrecordingmethods,comparingthesewithMAPassessmentsofactionpotentialwaveformdurationsandlatencies.
Ourmulti-electrodearraystudieswerelessextensive(550vs300μm;64vs247channels;1-vs2–4-kHzsampling)thanthoseusedpreviously[25].
Thesefindingswerethencorrelatedwithdifferencesinlevelsoffibrosis,thoughtheydidnotexploreforchangesinconnexinandNa+channelexpression.
Nevertheless,theycomplementedourearlierstudiesinexploringforinteract-ingeffectsofgenotype,age,andsexandpermittedanovelstatisticalanalysisofactivationtimesandtheirdispersionsbothovertheregionsofintereststudiedandthroughsuccessiveintrinsicbeats.
Stratificationoftheseresultsintoexperimentalgroupsparalleledandthereforepermitteddirectcomparisonswiththeearlier,similarlystratified,ECGresults[7],thusaddingtotheseafullclarificationofalterationsinelectrogramlatenciesatthetissuelevel.
Theywentfurthertoachievedirectcorrelationsofsuchalteredconductionwithquantitativeassessmentsoftheleveloffibrosis.
Thelattersuggestedpossiblemechanismsforsuchconductionchanges,therebyextendingandcomplementingearlierfindingsthathadalsoassessedforpossibleexistenceoffibrosis[10,17].
Thepresentexperimentsaccordinglyprovidedamoredetailedanalysisoftheseconductiondisordersonamoremicroscopicscaleasreflectedintheactivationtimesshownbyepicardialexcitationduringmulti-electrodearrayrecording.
Theysystematicallyexaminedtheeffectsofgenotype,age,andsex,correspondingtoeightexperimentalgroups,oncardiacconduction,determinedhowtheseeffectsalterthesubsequentdispersions,andrelatedthesefindingstomorpho-metricanalysisoffibrosistotestthisasapossiblemechanismunderlyingsuchelectrophysiologicalchanges.
Initially,thetimecourseofmonophasicactionpotentialrecoverywasassessedat30%,50%,70%,and90%recovery.
Thisyieldedsimilarvaluesthroughthestratifica-tionsindicatedabove,suggestingasimilarityinalltheirrecoverycharacteristics.
Arecentreport[11]haddescribedrightepicardialAPDsinScn5a+/thatdifferedfromWT.
However,thisonlyconsideredasingleageofmousebetween4and8months.
Itdidnotperformfurtherstratificationsbysex,incontrasttothepresentstudy.
Thisdifferenceinfindingsbetween3and~6monthssuggeststhatAPDcanvarywithage.
SuchanotionwouldparallelrecentdescriptionsofatrialremodelingphenomenainthepresenceofScn5amodification[6]andmeritsfurtherinvestigation.
Nevertheless,thepresentfindingsdodemon-stratedifferingactionpotentiallatenciescorroboratingearlierECGfindings[7]undercircumstancesinwhichactionpotentialcharacteristicsremainedsimilar.
OurinitialqualitativeassessmentofactivationmapsderivedfromactivationtimesateachchanneldemonstratedthattheScn5a+/groupgenerallyshowedhigheractivationtimescomparedwithWT,particularlytheoldermales.
Theyalsoshowedamoreheterogenouspatternofactiva-tion,attimeswithmultiplerandomfiringpoints.
Suchrandomactivationsuggeststhatthespreadofelectricalactivityintherightventricleisfragmented,conceivablyreflectingpatternsofspreadsuggestedonrecentoccasions[3].
Inthisrespect,itwouldresemblerecentreportsdescribingfragmentedQRScomplexescharacteristicsinTable7PercentageoffibrotictissueinmurineheartsAnimalSamplesAverage±SEWT234.
73±0.
80aWTyoung132.
18±0.
26b,cWTold107.
27±1.
08b,dWTmale124.
50±1.
02eWTfemale114.
95±1.
27WTyoungmale72.
22±0.
41f,gWTyoungfemale62.
12±0.
33hWToldmale57.
93±0.
95f,iWToldfemale56.
84±1.
75Scn5a+/2114.
38±2.
06aScn5a+/young119.
26±0.
93c,jScn5a+/old1018.
92±3.
14d,jScn5a+/male1118.
42±2.
82e,kScn5a+/female108.
60±1.
00kScn5a+/youngmale68.
78±1.
40g,lScn5a+/youngfemale59.
74±1.
37hScn5a+/oldmale524.
84±1.
53i,l,mScn5a+/oldfemale57.
08±1.
08mMeanswithsimilarlowercaselettersdiffersignificantlyfromeachotheratP<0.
05PflugersArch-EurJPhysiol(2011)461:29–4441BrSpatients[13].
ItfurthersuggeststhepresenceofareaswithintherightventriclethatdepolarizelaterthantherestparticularlyintheScn5a+/mice.
Suchafindingiscompatiblewiththelatepotentials,whichhavepreviouslybeenobservedinsignal-averagedECGthoughttorepresentregionsundergoingdelayeddepolarizationthantheremain-ingventriculartissue[27].
OtherclinicalstudieshavepreviouslyidentifiedthepresenceofsimilarlyheterogeneouspatternsinBrSpatients[9].
Inthisstudy,ratherthansuggestingasequentialspreadofconductionwithintheplaneoftheepicardium,theScn5a+/micesuggestedanirregularpatternofactivationinwhicheachepicardialsitewasseparatelyactivatedbyaradialpropagationofexcitationthroughthemyocardium.
Thesepatternswouldbecompatiblewithagreaterlikelihoodofre-entranteventsbetweenneighboringepicar-dialsitesandtherebyresultinarrhythmicsubstrate.
Thiswouldbeparticularlythecaseifneighboringsitesshowedstronglydifferentactivationtimes.
Thesemightbedetect-ablethroughthemaximumactivationtimesobservedandmighteventhenserveasanarrhythmictriggerinparallelwiththearrhythmogenicityobservedinthecorrespondingclinicalconditions.
Quantitativeassessmentscomparedsuchpatternsintermsofoverallactivationtimesandtheirdispersions,forthefirsttime.
Theseinturndemonstrateddifferencesthatwereattributabletobothindependentandinteractingeffectsofgenotype,age,andsex.
Firstly,ameanactivationtime(T*MEAN)providedanoverallrepresentationofepicardialactivationpropertiesresultingfrommyocardialconduction.
ThiswassignificantlyhigherintheScn5a+/comparedwithWThearts.
However,suchdifferenceswerelessclearwithfurtherstratificationbybothgenotypeandage.
Nevertheless,stratificationbybothgenotypeandsexresultedinhigherT*MEANvaluesinScn5a+/malecomparedwithScn5a+/femalehearts.
Thisestablishedaninteractingeffectofgenotypeandsexonconductiondisordersinmutationcarriers.
Thus,sexaffectsT*MEANvaluesonlyinthemutantandnotintheWT.
Thefinalstratificationinvolvinggenotype,age,andsexfurtherestablishedinteractingeffectsonT*MEANvaluesparticularlywithintheScn5a+/group.
Takentogether,thisanalysisdemonstratedthatalterationsinT*MEANvalues,likelyreflectingalteredconduction,areamajorfeatureofScn5a+/heartsparticularlyinoldmalehearts.
Theyalsodemonstrat-edthattheearlierelectrocardiographicfindings[7],sugges-tiveofalterationsinoverallconduction,furtherextendedtothepatternofepicardiumactivation.
SuchafindinginthemurineScn5a+/modelreflectsrecentclinicalfindingsimplicatingconductiondisorderasfeatureinarrythmogenicBrSpatients[15].
Secondly,atemporaldispersionD*Tdescribedthetemporalvariationinactivationtimeatepicardialsitesbetweencardiaccycles.
D*TvaluesweresignificantlyhigherintheScn5a+/hearts.
Althoughstratificationsbygenotypeandagedidnotyieldsignificantdifferences,thosebygenotypeandsexrevealeddifferencebetweenmaleScn5a+/andmaleWT.
Finally,stratificationbyallthreevariatesrevealeddifferencesbetweenoldmaleScn5a+/andoldmaleWT.
Takentogether,suchfindingssuggestthatwhereaninfluenceoccurs,temporaldispersionsoftheventricularactivationtimeareprimarilyaffectedbygenotypeandnotbyageandsex.
Thirdly,aspatialdispersion(D*S)describedthespatialvariationinactivationtimeofthedifferentepicardialsitesinasinglecardiaccycle.
UnlikebothT*MEANandD*T,valuesforD*Swereindistinguishablewhenstratifiedbygenotype,genotypeandage,andgenotypeandsex.
However,thefinalstratificationbygenotype,age,andsexyieldeddistinctsignificances.
Thus,itdemonstratedthattheoldmaleScn5a+/heartshadhigherdispersionsthannotonlytheoldmaleWTbutalsotheotherScn5a+/groups.
Suchafindingsuggeststhatdifferencesindispersionsofactivationtimesariseonlyfollowinginteractinginfluencesinvolvingallthreevariates.
Fourthly,themaximumactivationtime(T*MAX)de-scribedthefeaturesofthoseepicardialsitesshowingthegreatestactivationdelaysthatmightpotentiallyactuallytriggerarrhythmiaassuggestedabove.
T*MAXvaluesweresignificantlyhigherintheScn5a+/comparedwithWThearts,thusestablishinganindependenteffectofgenotypeonthisvalue.
Stratificationbygenotypeandageshowedmarkedeffects.
TheyrevealedhigherT*MAXvaluesinyoungScn5a+/thanyoungWT,inoldScn5a+/thanoldWT,andinoldScn5a+/thanyoungScn5a+/.
StratificationbygenotypeandsexrevealedhigherT*MAXvaluesinmaleScn5a+/thanmaleWT.
Finally,stratificationbyallthreevariatesrevealedstronglyinteractingeffectsspecificallyintheScn5a+/andnottheWT.
ThisresultedinhigherT*MAXvaluesinoldmaleScn5a+/comparedwitholdmaleWT,oldfemaleScn5a+/,andyoungmaleScn5a+/.
TheD*T,D*S,andT*MAXfindingsthussuggestedsignificantdifferencesindispersionsofepicardialactiva-tiontimesbetweenparticularexperimentalgroups.
Thefinalanalysisassessedtheextenttowhichthesedifferencescouldbeattributedtovariationsinconductionasrepre-sentedbyT*MEAN.
Thiswouldextendtheelectrocardio-graphicobservationsinourpreviouspaper[7]withthephysiologicalfindingsatthetissueleveldescribedhere.
SuchanotionwouldbecompatiblewithourstatisticalobservationsinwhichsignificantchangesinD*TandD*SwerealwaysaccompaniedbycorrespondingchangesinT*MEAN,butwiththereversenotbeingalwaysthecase.
Accordingly,weexploredforcorrelationsbetweentheabovemeasuresofdispersionwithsuchactivationtimes.
Thisdemonstratedlinearcorrelationsbetweeneachofthevariables,D*T,D*S,andT*MAXuponT*MEAN.
Eachgave42PflugersArch-EurJPhysiol(2011)461:29–44statisticallysignificantlinearcorrelationswhethertheexperimentalpopulationsweresortedbygenotype,age,orsex.
Furthermore,thisyieldedstatisticallyindistinguishableslopes,suggestingthatthesecorrelationsweresimilarthroughalltheseconditions.
Thisanalysisisthuscompat-iblewithasituationinwhichconductionpropertiesformthemajordeterminantforthesedispersions,regardlessofgenotype,age,andsex.
Afinalseriesofinvestigationstestedahypothesisthatimplicatedthedegreeoffibrosisintheconductionpropertiesfoundineachexperimentalgroup.
Theycom-plementedpreviousqualitativereportsthathadnotfullyassessedeffectsofbothageandsexinconnectionwithfibroticchangesassociatedwiththeScn5a+/mutation[10,17].
TheScn5a+/heartsthenshowedgenerallygreaterfibrosisthanWThearts.
Furthermore,evenyoungScn5a+/miceshowedincreasedfibrosisrelativetothecorrespondingyoungWT.
However,thispredispositionappearedtoapplyonlytothemaleandnotthefemaleScn5a+/mice.
Consequently,oldmaleScn5a+/showedgreaterfibrosisthantheyoungmaleScn5a+/,theoldfemaleScn5a+/,andtheoldmaleWThearts.
Thequantitativemorphometricanalysisofcollagenstainingusedherethusdemonstratedpatternsoffibrosisthatcloselymatchedtheelectrophysiologicalfindingsamongtheexperimentalgroupssortedbygenotype,age,andsex.
Insummary,wehaveinvestigatedforindependentandinteractingeffectsoftheScn5a+/genotype,age,andsexonepicardialactivationanditsdispersioninmurinehearts,whichhavebeenpreviouslyusedtomodelthecorrespondinghumanclinicalconditions.
Ourfindingsshowthatgenotype,age,andsexallexertindependentandinteractingeffectsoncardiacconductionwiththegreatesteffectsonventricularactivationtimesintheoldmaleScn5a+/.
Suchchangesdirectlycorrelatedwiththecorrespondinglevelsoffibrosis,therebydirectlyimplicat-ingfibroticchangesasimportantparticipantsintheseconductiondifferences.
Theseeffectstookplacedespiteanabsenceofcorrespondingdifferencesinactionpotentialrecoverytimesasreflectedinactionpotentialdurationsmeasuredat30%,50%,70%,and90%fullrecovery.
TheseeffectsonconductioncorrelatedwithgreaterdispersionsofactivationtimesintheScn5a+/heartsasawholecomparedwithWT.
Theinvolvedvariationsinactivationtimearebothbetweencardiaccyclesatanygivenrecordingsiteandbetweenrecordingsiteswithinagivencardiaccycle,aswellasthemaximumactivationtimesthatwereobtainedduringsuchrecordings.
Thesedispersionshavebeenimplicatedinarrhythmicsubstrate,asassessedbyelectro-gramanalysisinclinicalstudies[19].
InmurineScn5a+/,theincreasedEGDthathadbeenreportedpreviously[23]aredirectlycomparablewiththeincreaseddispersioninepicardialactivationreportedhere.
ThisfindingagainwasparticularlymarkedinheartsfromoldmaleScn5a+/mice.
Thesubsequentregressionanalysisgaveregressioncoef-ficientsthatattributedeachofthesedispersionsdirectlytodifferencesinconduction.
Ineachcase,therewasdepen-dencebetweenvariates,m,thatwassimilarthroughallthestratificationsthattheexperimentsexplored.
Suchfindingsimplicatesimilaractionsofgenotype,age,orsexuponeachdispersion,whicharemediatedthroughactionsontheconductionprocess.
Together,thesefindingsalsodemon-stratethatincontrasttotheirsimilarepicardialrepolariza-tionfeatures,conductionanddispersion,whicharefeaturesofdepolarization,areprominentinthemurineScn5a+/model,particularlyinoldermales.
Theymaythuscontrib-utetothereportedarrhythmogenicpropertiesofthissystem.
AcknowledgementsKamalanJeevaratnamissupportedbytheMaxisScholarshipforExcellenceProgram,fundedandadministeredbyMaxisCommunicationsBerhad(Malaysia).
HeisalsotherecipientoftheCambridgeCommonwealthTrustBursaryAwardandtheLundgrenResearchAward.
RebeccaRewburywassupportedbyaWellcomeTrustSummerResearchStudentship.
ThisresearchisfundedbytheBritishHeartFoundation,theMedicalResearchCouncil,andtheWellcomeTrust,UnitedKingdom.
WethankProf.
WimLammersoftheFacultyofMedicine,UAEUniversity,forhelpinthedesignandconceptionofthemulti-arrayunitandtoDowningCollegeandtheAvrithGrantforconferencesupport.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttributionNoncommercialLicensewhichper-mitsanynoncommercialuse,distribution,andreproductioninanymedium,providedtheoriginalauthor(s)andsourcearecredited.
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